Filaggrin gene expression promoter

ABSTRACT

The present invention provides a novel substance that promotes expression of filaggrin gene involved in improvement of the moisture retention function of skin. The inventors of the present invention found that expression of filaggrin gene fluctuates rhythmically over a roughly 24 hour cycle, screened candidate substances based on the time at which expression reaches a maximum following addition of the candidate substance, and identified zanthoxylum extract, 3-(1′-piperidine)propionic acid, geranium oil, cypress oil, rose oil, galvanum oil, pepper oil, basil oil, methyl-o-toluate, methyl anthranilate and dimethyl anthranilate as filaggrin gene expression promoting agents.

TECHNICAL FIELD

The present invention relates to a cosmetic and external skinpreparation, and more particularly, to a promoting agent of theexpression of filaggrin gene that is involved in improvement of themoisture retention function of skin.

BACKGROUND ART

The horny layer of the skin covers the living body whereby retainingmoisture and providing a defense against invasion by foreign substances.The horny layer is composed of various substances such as proteinsincluding keratin and filaggrin, and lipids, produced by epidermalkeratinocytes. Filaggrin fulfills the important role of forming astructure referred to as a keratin pattern by assembling keratin fibersinto layers. Filaggrin is produced from profilaggrin, a precursorencoded by filaggrin gene (FLG), accompanying with keratinization, andcauses agglomeration of keratin fibers. Moreover, filaggrin isdecomposed in the upper corneal layer, thereby it is transformed intolow molecular weight peptides and amino acids referred to as naturalmoisturizing factors, and is involved in moisture retention andabsorption of ultraviolet rays.

In this manner, filaggrin is intimately involved in the moistureretention and barrier function of the horny layer of the skin, and adecrease in filaggrin production is involved in the onset of dry skindiseases such as ichthyosis vulgaris, atopic dermatitis or senilexerosis (Non-Patent Document 1). In order to maintain healthy andattractive skin condition, the suitable production of filaggrin and asuitable cycle of continuous cornification are required.

In consideration of the above, research has been conducted on substancesthat promote filaggrin synthesis, and plant extracts such as wild thymeextract, clove extract, salvia extract, etc., are already known to besubstances having effects that promote filaggrin synthesis (PatentDocument 1). In addition, ectoin, which is accumulated by certainspecies of bacteria in order to regulate osmotic pressure, is known toinhibit decreases in filaggrin caused by drying (Patent Document 2).

PRIOR ART DOCUMENTS Patent Documents

Patent Document 1: Japanese Patent No. 4768238

Patent Document 2: Japanese Patent No. 4540872

Non-Patent Documents

Non-Patent Document 1: Morar, N., et al., Filaggrin mutations inchildren with severe atopic dermatitis, Journal of InvestigativeDermatology, 127, 1667-1672, 2007

Non-Patent Document 2: Akashi, M., et al., Noninvasive method forassessing the human circadian clock using hair follicle cells, Proc.Natl. Acad. Sci. USA, 107(35), 15643-15648, 2010

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

As was previously described, there is a strong desire among consumersfor a substance having a more potent effect of promoting filaggrinsynthesis, and development of a novel filaggrin synthesis promotingagent is currently sought. Thus, a problem to be solved by the presentinvention is to provide a novel substance that promotes filaggrin geneexpression.

Means for Solving the Problems

As a result of conducting extensive studies on the expression offilaggrin gene, the inventors of the present invention surprisinglyfound for the first time that expression of filaggrin gene fluctuatesrhythmically in a roughly 24 hour cycle. Thus, it was found that, inorder to promote filaggrin production and maintain the skin in a healthystate, in addition to simply enhancing filaggrin production orinhibiting decreases in filaggrin production caused by drying as wascarried out in the prior art, it is also important to promote productionof filaggrin in consideration of this rhythmical property. Therefore,candidate substances were screened for filaggrin gene expressionpromoting action in consideration of this rhythmical property, therebyleading to completion of the filaggrin gene expression promoting agentof the present invention.

Zanthoxylum extract, 3-(1′-piperidine)propionic acid, geranium oil,cypress oil, rose oil, galvanum oil, pepper oil, basil oil,methyl-o-toluate, methyl anthranilate and dimethyl anthranilate, wereselected by screening carried out by the inventors of the presentinvention as an agent having an effect of promoting filaggrin geneexpression.

Thus, the present invention relates to a filaggrin gene expressionpromoting agent containing one or more substances selected from thegroup consisting of zanthoxylum extract, 3-(1′-piperidine) propionicacid, geranium oil, cypress oil, rose oil, galvanum oil, pepper oil,basil oil, methyl-o-toluate, methyl anthranilate and dimethylanthranilate.

In another aspect of the present invention, the present inventionrelates to a method for promoting expression of filaggrin genecomprising administering one or more filaggrin gene expression promotingagents selected from the group consisting of zanthoxylum extract,3-(1′-piperidine)propionic acid, geranium oil, cypress oil, rose oil,galvanum oil, pepper oil, basil oil, methyl-o-toluate, methylanthranilate and dimethyl anthranilate to a subject requiring promotionof the expression of filaggrin gene.

In another aspect of the present invention, the present inventionrelates to use of one or more substances selected from the groupconsisting of zanthoxylum extract, 3-(1′-piperidine)propionic acid,geranium oil, cypress oil, rose oil, galvanum oil, pepper oil, basiloil, methyl-o-toluate, methyl anthranilate and dimethyl anthranilate forpreparing a filaggrin gene expression promoting agent.

Moreover, the present invention relates to a method for promotingexpression of filaggrin gene or a cosmetic method comprisingadministering a filaggrin gene expression promoting agent at a time atwhich the biological rhythm of filaggrin gene expression level and therhythm of the filaggrin gene expression level resulting fromadministration of the filaggrin gene expression promoting agent aresynchronous, based on the finding that expression of filaggrin genefluctuates rhythmically in a roughly 24 hour cycle. Moreover, thepresent invention also relates to a filaggrin gene expression promotingagent that is used such that the filaggrin gene expression promotingagent is administered at a time at which the biological rhythm offilaggrin gene expression level and the rhythm of the filaggrin geneexpression level resulting from administration of the filaggrin geneexpression promoting agent are synchronous, and to a filaggrin geneexpression promoting agent that is used such that the filaggrin geneexpression promoting agent is administered at a time at which the rhythmof the filaggrin gene expression level is restored by administration ofthe filaggrin gene expression promoting agent in a subject in which thebiological rhythm of filaggrin gene expression level is disturbed.

Effects of the Invention

The filaggrin gene expression promoting agent according to the presentinvention has at least one of the effects listed below:

promotion of the expression of filaggrin gene;

enhancement of the skin's barrier function; and

moisture retention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph indicating the rhythmic 24-hour fluctuations offilaggrin gene expression in cultured keratinocytes temporarily culturedin medium containing cortisol.

FIG. 2 is a graph indicating the rhythmic 24-hour fluctuations in theexpression levels of filaggrin gene and clock genes BMAL1 and PER3 incultured keratinocytes temporarily cultured in medium containingcortisol.

FIG. 3 is a bar graph indicating the results for filaggrin geneexpression promoting action of candidate pharmaceutical agents.

FIG. 4 is a bar graph indicating the results for filaggrin geneexpression promoting action in the case of using3-(1′-piperidine)propionic acid, zanthoxylum extract and a combinationthereof as candidate pharmaceutical agents.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a filaggrin gene expression promotingagent comprising one or more substances selected from the groupconsisting of zanthoxylum extract, 3-(1′-piperidine)propionic acid,geranium oil, cypress oil, rose oil, galvanum oil, pepper oil, basiloil, methyl-o-toluate, methyl anthranilate and dimethyl anthranilate.The substances exemplified above may be used alone for the filaggringene expression promoting agent or may be used in combination. Morepreferably, zanthoxylum extract is selected for the filaggrin geneexpression promoting agent of the present invention. Even morepreferably, a combination of zanthoxylum extract and3-(1′-piperidine)propionic acid is selected for the filaggrin geneexpression promoting agent.

In another aspect thereof, the filaggrin gene expression promoting agentof the present invention further comprises another previously knownpharmaceutical agent having a filaggrin gene expression promoting effectsuch as wild thyme extract, clove extract, salvia extract or ectoin.

The filaggrin gene expression promoting agent of the present inventionis able to promote filaggrin synthesis by promoting expression offilaggrin gene. More specifically, profilaggrin is synthesized throughthe expression of filaggrin gene, is subjected to phosphorylation, andthen is subjected to dephosphorylation and hydrolysis by peptidylarginine deiminase during keratinization, thereby filaggrin issynthesized. Thus, a filaggrin gene expression promoting agent is alsoreferred to as a profilaggrin gene expression promoting agent, and mayalso be a filaggrin synthesis promoting agent that promotes synthesis offilaggrin by promoting expression of filaggrin gene.

The filaggrin gene expression promoting agent of the present inventionpromotes filaggrin synthesis and further brings about an increase inamino acids that are the main components of natural moisturizing factors(NMF) produced from the decomposition of filaggrin. Thus, the filaggringene expression promoting agent of the present invention may also be anatural moisturizing factor enhancing agent, a moisturizing agent, orskin barrier function enhancing agent, since it enhances naturalmoisturizing factors, and is incorporated in a cosmetic. Moreover, dryskin and dry skin diseases such as senile xerosis, atopic dermatitis orichthyosis vulgaris can be treated by the filaggrin gene expressionpromoting agent or method for promoting expression of filaggrin gene ofthe present invention.

In one aspect of the present invention, the filaggrin gene expressionpromoting agent of the present invention may be administered through anarbitrary route, and may be administered either orally or parenterally(such as by transcutaneous, transmucosal, subcutaneous, intravenous,intraperitoneal, transpulmonary or intramuscular administration). In thecase of oral administration, the filaggrin gene expression promotingagent may be administered by formulating into a tablet or capsule andthe like, or may be administered as a drink or supplement. In the caseof transcutaneous administration, it may be incorporated in a cosmeticor external skin preparation and may be administered by applying to theskin. In the case of transpulmonary administration, the filaggrin geneexpression promoting agent may be administered by inhaling a mist of asolution obtained by the dissolution thereof or inhaling aftervaporizing, and may be administered in the form of aroma therapy, forexample. The filaggrin gene expression promoting agent is preferablyadministered transcutaneously from the viewpoint of promoting expressionof filaggrin gene in epidermal cells.

The filaggrin gene expression promoting agent of the present inventionis administered to not only subjects requiring promotion of expressionof filaggrin gene, such as subjects desiring enhancement of moisturizingand the skin's barrier function, but also to subjects in which thebiological rhythm of filaggrin gene expression level is disturbed. Thesubjects requiring promotion of filaggrin gene expression include, forexample, subjects suffering from dry skiun, dry skin diseases such assenile xerosis, atopic dermatitis or ichthyosis vulgaris.

The filaggrin gene expression promoting agent of the present inventionrefers to a cosmetic, pharmaceutical, quasi-drug or product applied tothe skin, and therefore the form thereof can be any of a wide range offorms such as an aqueous solution type, solubilized type, emulsifiedtype, powdered type, gel type, ointment type, cream, water-oil two-layertype or water-oil-powder three-layer type. Examples of cosmetics towhich the present invention can be applied include skin lotions, creams,milky lotions, gel, beauty essence, ointments, packs, bath additives,body soaps, shampoos, rinses and foundations. In the case of apharmaceutical or quasi-drug, the present invention can be applied invarious forms such as ointments or creams.

The cosmetic and/or external skin preparation of the present inventionmay also contain active ingredients and/or excipients commonly used inthe production of cosmetics and/or external skin preparations, andexamples thereof include bases such as water, alcohol, glycerin orhyaluronic acid, surfactants, moisturizers, thickeners, pH adjusters, UVabsorbing agent, stabilizers, antimicrobials, fragrances and so on.

As shown in FIG. 1, expression of filaggrin gene rhythmically fluctuatesover a roughly 24 hour cycle in a cultured cell system of keratinocytes,and this rhythm is thought to be equivalent to circadian rhythm.Circadian rhythm is a cyclical rhythm having a roughly 24 hour cyclethat is controlled by a feedback loop composed of clock proteins such asCLOCK, BMAL1, CRY or PER, and is known to have an effect on variousphysiological phenomena such as sleep/awakedness, hormone secretion,body temperature and cell cycle in the body.

Since expression levels of filaggrin gene are thought to fluctuate inline with circadian rhythm in vivo, the filaggrin gene expressionpromoting agent of the present invention can promote fillaggrinsynthesis more effectively by administering the filaggrin geneexpression promoting agent in conformance with circadian rhythm, andmore specifically, by administering so as to synchronize with the timeof the maximum or minimum expression level of filaggrin expression.

In another aspect of the present invention, the present inventionrelates to a cosmetic method comprising administering a filaggrin geneexpression promoting agent at a time at which the biological rhythm offilaggrin gene expression level and the rhythm of the filaggrin geneexpression level resulting from administration of the filaggrin geneexpression promoting agent are synchronous. In the present invention, acosmetic method refers not only to a method carried out by anindividual, but also refers to that provided as a cosmetic formula inaccordance with customer preferences when providing beauty products aswell as that provided by a cosmetic salesperson or esthetician otherthan a physician.

In another aspect of the present invention, the present inventionrelates to a filaggrin gene expression promoting agent that is used soas to be administered at a time at which the biological rhythm offilaggrin gene expression level and the rhythm of the filaggrin geneexpression level resulting from administration of the filaggrin geneexpression promoting agent are synchronous.

In still another aspect, the present invention relates to a method forpromoting expression of filaggrin gene that comprises administering afilaggrin gene expression promoting agent at a time at which thebiological rhythm of filaggrin gene expression level and the rhythm ofthe filaggrin gene expression level resulting from administration of thefilaggrin gene expression promoting agent are synchronous in a subjectrequiring promotion of filaggrin gene expression. This method forpromoting expression of filaggrin gene is a method for treating dry skinand dry skin diseases such as senile xerosis, atopic dermatitis orichthyosis vulgaris.

Synchronous administration refers to administering such that the peak ofthe biological rhythm of filaggrin gene expression level and the peak offluctuations in filaggrin gene expression level resulting fromadministration of the filaggrin gene expression promoting agent overlap,and refers to the administration of filaggrin gene expression promotingagent at 16 hours to 22 hours, more specifically 17 hours to 21 hours,and even more specifically 18 hours to 20 hours, prior to the timefilaggrin gene expression level reaches a maximum.

In actuality, since the peak of filaggrin gene expression in humans isestimated to be from about 5:00 AM to 11:00 AM, more specifically fromabout 6:00 AM to 10:00 AM, and even more specifically from about 7:00 AMto 9:00 AM, the filaggrin gene expression promoting agent is thought tobe preferably administered, for example, at 16 hours to 22 hours, morespecifically 17 hours to 21 hours, and even more specifically 18 hoursto 20 hours prior thereto. For example, the filaggrin gene expressionpromoting agent is thought to be preferably administered between thetimes of about 7:00 AM to 1:00 PM to about 1:00 PM to 7:00 PMcorresponding to 16 hours to 22 hours prior to the peak of filaggringene expression.

As was clearly determined in the present invention, filaggrin isexpressed in accordance with the rhythm of the body's internal clock,and when this rhythm of the body's internal clock is disturbed by someform of cause such as aging, stress, inadequate sleep or disturbances indaily rhythm, the rhythm of filaggrin expression is thought to shiftcorrespondingly or the expression level thereof is thought to decrease.Thus, by administering the filaggrin gene expression promoting agent ofthe present invention at the proper timing, restoration of theexpression rhythm of filaggrin can be expected to be restored orexpression levels thereof can be expected to increase. Therefore, inanother aspect of the present invention, the filaggrin gene expressionpromoting agent of the present invention administered at the propertiming can be used as an agent for improving or restoring the expressionlevel of filaggrin. With respect to this proper timing, since the peakof filaggrin gene expression in humans is estimated to be from about5:00 AM to 11:00 AM, more specifically from about 6:00 AM to 10:00 AM,and even more specifically from about 7:00 AM to 9:00 AM, the filaggringene expression promoting agent is thought to be preferably administered16 hours to 22 hours, more specifically 17 hours to 21 hours, and evenmore specifically 18 hours to 20 hours prior thereto.

Since the expression of filaggrin gene fluctuates cyclically roughlyevery 24 hours, when screening filaggrin gene expression promotingagents, by comparing filaggrin gene expression level during the time ofminimum expression roughly 0 hours to 10 hours, preferably 2 hours to 8hours and more preferably 4 hours to 6 hours after administering acandidate pharmaceutical agent to cultured cells such as culturedkeratinocytes, with filaggrin gene expression level during the time ofmaximum expression roughly 16 hours to 22 hours, preferably 17 hours to21 hours and more preferably 18 hours to 20 hours after administeringthe candidate pharmaceutical agent, filaggrin gene expression promotingagents can be screened more accurately. In still another aspect,filaggrin gene expression promoting agents can be screened by usingfilaggrin gene expression level during the time of maximum expression asan indicator. In still another aspect, filaggrin gene expressionpromoting agents can be screened by determining fillaglin geneexpression level at the time of maximum expression after the secondcycle, such as at 40 hours to 48 hours or at 64 hours to 72 hours, as anindicator.

Geranium oil, which is one of the pharmaceutical agents thatdemonstrates the effect of the filaggrin gene expression promoting agentof the present invention, is an essential oil derived from the entireplant of a plant belonging to the genus Pelargonium such as Pelargoniumgraveolens, and examples thereof include essential oils obtained bysteam distillation of the entire plant of plants belonging to theaforementioned genus Pelargonium.

Cypress oil, which is one of the pharmaceutical agents that demonstratesthe effect of the filaggrin gene expression promoting agent of thepresent invention, is an essential oil derived from Cupressussempervirens, and examples thereof include essential oils obtained bysteam distillation of the branches and leaves of Cypress plants.

Rose oil, which is one of the pharmaceutical agents that demonstratesthe effect of the filaggrin gene expression promoting agent of thepresent invention, is an essential oil derived from the flower petals ofa plant belonging to the genus Rosa such as Rosa centifolia L. or Rosadamascene Mill., and examples thereof include essential oils obtained bysteam distillation of the flower petals of plants belonging to theaforementioned genus Rosa.

Galvanum oil, which is one of the pharmaceutical agents thatdemonstrates the effect of the filaggrin gene expression promoting agentof the present invention, is an essential oil derived from a rubberyexudate that seeps from the leaves and buds of Ferula galbaniflua of thefamily Umbelliferae and related species thereof, and examples thereofinclude essential oils obtained by steam distillation.

Pepper oil, which is one of the pharmaceutical agents that demonstratesthe effect of the filaggrin gene expression promoting agent of thepresent invention, is an essential oil derived from the berries ofpepper (Piper nigrum L.), a perennial plant belonging to the familyPiperaceae, and examples thereof include essential oils obtained bysteam distillation of the berries thereof.

Basil oil, which is one of the pharmaceutical agents that demonstratesthe effect of the filaggrin gene expression promoting agent of thepresent invention, is an essential oil derived from the entire aboveground portion of basil (Ocimum basilicum L.), an annual belonging tothe family Labiatae, and examples thereof include essential oilscollected by steam distillation of the entire above ground portionthereof.

Zanthoxylum extract, which is one of the pharmaceutical agents thatdemonstrates the effect of the filaggrin gene expression promoting agentof the present invention, is an extract of Zanthoxylum piperitumbelonging to the genus Zanthoxylum of the family Rutaceae. Thezanthoxylum extract of the present invention can be obtained inaccordance with ordinary methods, such as by immersing or heating toreflux a leaf, stem, root, flower, fruit, bark, stone or the entireplant of zanthoxylum, or mixture thereof, with an extraction solventfollowed by filtration and concentration. Any extraction solvent can beused provided it is ordinarily used for extraction, and examples ofextraction solvents include water, alcohols such as methanol, ethanol,propylene glycol, 1,3-butylene glycol or glycerin, aqueous alcohols, andorganic solvents such as chloroform, dichloroethane, carbontetrachloride, acetone, ethyl acetate or hexane, and these can each beused alone or used in combination. In addition, extracts obtained byextracting with the aforementioned solvents can be used directly, orconcentrated extracts can be adsorbed with a porous polymer column (suchas the Amberlite XAD-2) after having removed impurities using anadsorption method or ion exchange resin and the like, followed byeluting with methanol or ethanol and concentrating prior to use. Inaddition, an extract obtained by extracting using a partition method,such as a mixture of water and ethyl acetate, can also be used. Theextract is preferably extracted using a lowly irritative solvent such aswater, 1,3-butylene glycol or glycerin from the viewpoint of using in acosmetic or pharmaceutical such as an external skin preparation that isapplied directly to the skin.

An essential oil refers to a mixture of insoluble to poorly solubleorganic compounds in water contained in a plant, typically containsvolatile organic compounds, and may be aromatic according to the rawmaterial used. Since an essential oil is a mixture of insoluble topoorly soluble organic compounds in water contained in a plant, it istypically formed by steam distillation. However, in the presentinvention, an essential oil is not intended to be limited to a mixtureformed by steam distillation, but rather is intended to includewater-insoluble or poorly soluble extracts that use a portion of a plantbody as raw material and are extracted with an organic solvent. Namely,in the present invention, an “essential oil” refers to a mixture oforganic compounds specified according to the plant used as raw material(and the site thereof depending on the case) that demonstrate thedesired effect of the present invention, namely action that promotesexpression of filaggrin gene.

Methyl o-toluate (also known as methyl 2-methylbenzoate), which is oneof the pharmaceutical agents that demonstrates the effect of thefilaggrin gene expression promoting agent of the present invention, is acompound represented by the molecular formula C₉H₁₀O₂, has a molecularweight of 150.17, and is a liquid at normal temperature.

Methyl anthranilate (also known as methyl 2-aminobenzoate), which is oneof the pharmaceutical agents that demonstrates the effect of thefilaggrin gene expression promoting agent of the present invention, is acompound represented by the molecular formula C₈H₉O₂N, has a molecularweight of 151.17, and is a liquid at normal temperature.

Dimethyl anthranilate (also known as 2-methyl aminomethylbenzoate),which is one of the pharmaceutical agents that demonstrates the effectof the filaggrin gene expression promoting agent of the presentinvention, is a compound represented by the molecular formula C₉H₁₁O₂N,has a molecular weight of 165.20, and is a liquid at normal temperature.

3-(1′-piperidine)propionic acid (1PP), which is one of thepharmaceutical agents that demonstrates the effect of the filaggrin geneexpression promoting agent of the present invention, is a compoundrepresented by the molecular formula C₈H₁₅NO₂, has a molecular weight of157.21, and is a solid at normal temperature.

EXAMPLES Example 1 Examination of Filaggrin Gene Expression in HumanSkin-Derived Keratinocytes

Commercially available normal adult skin-derived keratinocytes (CellntecAG) were disseminated in a culture plate to a concentration of 3×10³cells/cm² followed by culturing in epithelial cell medium (CnT-BM.1,Cellntec AG) at 37° in a 5% CO₂ atmosphere. Three days later, the mediumwas replaced with medium containing cortisol at 50 ng/mL followed bysynchronously culturing at 37° C. and 5% CO₂. Two hours later, culturingwas continued after replacing the medium with ordinary medium afterwhich the cells were collected over time. RNA was extracted from thecollected cells using a commercially available RNA extraction-cDNAsynthesis kit (FastLane Cell cDNA Kit, Qiagen N.V.) to prepare cDNA.Filaggrin expression levels were measured by quantitative PCR (QPCR)using this cDNA. Expression levels of clock genes PER3 and BMAL1 weremeasured simultaneously and compared with the expression rhythm offilaggrin gene. A commercially available QPCR reagent kit (Brilliant IIIUltra-Fast SYBR Green QPCR Kit, Agilent Technologies Inc.) and QPCRmeasurement system (MX-3000P Real-time Quantitative PCR System, AgilentTechnologies Inc.) were used for QPCR. The expression level of ahousekeeping gene in the form of RPLP0 gene was quantified for use as aninternal standard, and the relative expression level with respect toPLP0 was calculated and used as the expression level of filaggrin gene.Commercially available filaggrin and PER3, BMAL1 and RPLP0 primers(Perfect Real Time Primer, Takara Bio Inc.) were used for the PCRprimers. The sequences of the primers used are shown in Table 1.

TABLE 1  Primer Sequences 5′→3′ Filaggrin Forward CTCAGGCACTGGGCGCAGACReverse GCCTGTCCGTGGGCTGACAC PER3 Forward ATGCGGTTACAGCAGCACCA ReverseAGGGTCCAGGGCTCACAGAA BMAL1 Forward CTCCAGGAGGCAAGAAGATTT ReverseCTACTTGATCCTTGGTCGTTG RPLPO Forward GGCGACCTGGAAGTCCAACT ReverseCCATCAGCACCACAGCCTTC

The results are shown in FIGS. 1 and 2. Expression of filaggrin gene wasfound to fluctuate rhythmically over a roughly 24 hour cycle in acultured cell system of keratinocytes, and in the keratinocytes, theexpression level of filaggrin gene was found to reach a maximum about 18hours after the start of synchronous culturing with cortisol. Thisrhythm is thought to be equivalent to circadian rhythm. In addition, theexpression rhythm of filaggrin gene was in the same phase as theexpression rhythm of clock gene PER3 but was in the opposite phase fromBMAL1. In actuality, the expression peak of PER3 in humans has beenreported to be from 5:00 AM to 11:00 AM, more specifically from 6:00 AMto 10:00 AM and even more specifically from 7:00 AM to 9:00 AM(Non-Patent Document 2). Based on a comparison between the peakexpression time of PER3 and the peak expression time of filaggrin geneas shown in FIG. 2, the expression peak of filaggrin gene and theexpression peak of PER3 in humans are presumed to be roughly the same.

Example 2 Screening Pharmaceutical Agents that Promote Filaggrin GeneExpression

Commercially available normal adult skin-derived keratinocytes (CellntecAG) were disseminated in a culture plate to a concentration of 3×10³cells/cm² followed by culturing in epithelial cell medium (CnT-BM.1,Cellntec AG) at 37° in a 5% CO₂ atmosphere. Three days later, the mediumwas replaced with medium containing each of the candidate pharmaceuticalagents at a concentration of 1% followed by continuing culturing at 37°C. and 5% CO₂ and collecting the cells after 2 hours and after 18 hours.The candidate pharmaceutical agents added to the medium consisted ofzanthoxylum extract (Maruzen Pharmaceuticals Co., Ltd.), geranium oil(Koei Kogyo Co., Ltd.), cypress oil (Bioland Co., Ltd.), rose oil(Bioland Co., Ltd.), galvanum oil (Bioland Co., Ltd.), pepper oil(Bioland Co., Ltd.), basil oil (Bioland Co., Ltd.), methyl-o-toluate(Aldrich Inc.), methyl anthranilate (Tokyo Chemical Industry Co., Ltd.),dimethyl anthranilate (Tokyo Chemical Industry Co., Ltd.), clove budcaryophyllata oil (Bioland Co., Ltd.) and arnica oil (Koei Kogyo Co.,Ltd.). The absence of addition of pharmaceutical agent was used as anegative control, and cortisol (concentration in medium: 50 ng/mL) wasused as a positive control.

RNA was extracted from the collected cells using a commerciallyavailable RNA extraction-cDNA synthesis kit (FastLane Cell cDNA Kit,Qiagen N.V.) to prepare cDNA. Filaggrin expression levels were measuredby quantitative PCR (QPCR) using this cDNA. Expression levels of clockgenes PER3 and BMAL1 were measured simultaneously and compared with theexpression rhythm of filaggrin gene. A commercially available QPCRreagent kit (Brilliant III Ultra-Fast SYBR Green QPCR Kit, AgilentTechnologies Inc.) and QPCR measurement system (MX-3000P Real-timeQuantitative PCR System, Agilent Technologies Inc.) were used for QPCR.The expression level of a housekeeping gene in the form of RPLP0 genewas quantified for use as an internal standard, and the relativeexpression level with respect to PLP0 was calculated and used as theexpression level of filaggrin gene. The primer sequences shown in Table1 were used for the sequences of the PCR primers.

FIG. 3 shows expression levels of filaggrin gene at 2 hours and 18 hoursafter addition of each of the candidate pharmaceutical agents.Zanthoxylum extract, geranium oil, cypress oil, rose oil, galvanum oil,pepper oil, basil oil, methyl-o-toluate, methyl anthranilate anddimethyl anthranilate were shown to promote expression of filaggrin geneafter 18 hours in comparison with the negative control. On the otherhand, although expression of filaggrin gene increased after 18 hours inthe case of clove bud and arnica, there were no significant differencesbetween the amounts of those increases and the control.

Example 3 Pharmaceutical Agents Promoting Expression of Filaggrin Gene

Commercially available normal adult skin-derived keratinocytes (CellntecAG) were disseminated in a culture plate to a concentration of 3×10³cells/cm² followed by culturing in epithelial cell medium (CnT-BM.1,Cellntec AG) at 37° in a 5% CO₂ atmosphere. Three days later, the mediumwas replaced with medium containing 3-(1′-piperidine)propionic acid(1PP, Yuki Gousei Kogyo Co., Ltd.) at a concentration of 0.1%, mediumcontaining zanthoxylum extract at a concentration of 0.1%, and mediumcontaining both 1PP at 0.1% and zanthoxylum extract at 0.1%, followed bycontinuing culturing at 37° C. and 5% CO₂ and collecting the cells after18 hours. The absence of addition of pharmaceutical agent was used as acontrol.

RNA was extracted from the collected cells using a commerciallyavailable RNA extraction-cDNA synthesis kit (FastLane Cell cDNA Kit,Qiagen N.V.) to prepare cDNA. Filaggrin expression levels were measuredby quantitative PCR (QPCR) using this cDNA. A commercially availableQPCR reagent kit (Brilliant III Ultra-Fast SYBR Green QPCR Kit, AgilentTechnologies Inc.) and QPCR measurement system (MX-3000P Real-timeQuantitative PCR System, Agilent Technologies Inc.) were used for QPCR.The expression level of a housekeeping gene in the form of RPLP0 genewas quantified for use as an internal standard, and the relativeexpression level with respect to PLP0 was calculated and used as theexpression level of filaggrin gene. The primer sequences shown in Table1 were used for the sequences of the PCR primers. The results are shownin FIG. 4. 1PP at a concentration of 0.1% demonstrated an effect ofpromoting filaggrin gene expression that was comparable to that ofzanthoxylum extract at 0.1%, while the combined use of zanthoxylumextract and 1PP further promoted expression of filaggrin gene.

Formulation Examples

Although the following indicates formulation examples of the filaggringene expression promoting agent of the present invention, working of thepresent invention is not limited to the following examples.

Fragrance

(1) Alcohol 75.0 (2) Purified water Balance (3) Dipropylene glycol  5.0(4) Filaggrin gene expression promoting agent of 10.0 present invention:Rose oil (5) Antioxdiant  8.0 (6) Pigment As suitable (7) Ultravioletabsorber As suitable

Room Fragrance

(1) Alcohol 80.0  (2) Purified water Balance (3) Antioxidant 5.0 (4)Filaggrin gene expression promoting agent of 3.0 present invention:Cypress oil (5) 3-methyl-3-methoxybutanol 5.0 (6) Dibenzylidene sorbitol5.0

Incense

(1) Tabunoki powder 75.5 (2) Sodium benzoate 15.5 (3) Filaggrim geneexpression promoting agent of 5.0 present invention: Galvanum oil (4)Eucalyptus oil 1.0 (5) Purified water Balance

Bath Additive

(1) Sodium sulfate 45.0 (2) Sodium bicarbonate 45.0 (3) Lavender oil 9.0(4) Filaggrin gene expression promoting agent of 1.0 present invention:Methyl anthranilate

Massage Gel

(1) Erythritol 2.0 (2) Caffeine 5.0 (3) Phellodendron bark extract 3.0(4) Glycerin 50.0 (5) Carboxyvinyl polymer 0.4 (6) Polyethylene glycol400 30.0 (7) Trisodium edetate 0.1 (8)Polyoxylene(10)-methylpolysiloxane 2.0 copolymer (9) Squalane 1.0 (10)Potassium hydroxide 0.15 (11) Filaggrin gene expression promoting agent1.0 of present invention: Geranium oil

Massage Cream

(1) Solid paraffin 5.0 (2) Beeswax 10.0 (3) Vaseline 15.0 (4) Liquidparaffin 41.0 (5) 1,3-butylene glycol 4.0 (6) Glycerin monostearate 2.0(7) POE(20) sorbitan monolaurate 2.0 (8) Borax 0.2 (9) Caffeine 2.0 (10)Antiseptic As suitable (11) Antioxidant As suitable (12) Filaggrin geneexpression promoting agent 1.0 of present invention: Zanthoxylum extract(13) Purified water Balance

Aromatic Fibers

Microcapsules containing the filaggrin gene expression promoting agentof the present invention (particle diameter: 50 μm or less, ratio ofpharmaceutical agent in microcapsule: 50% by weight) were added to andmixed with a cuprammonium cellulose solution (cellulose concentration:10% by weight, ammonium concentration: 7% by weight, copperconcentration: 3.6% by weight) within the range of 1% to 20% based onthe weight of cellulose followed by spinning into fibers in accordancewith an ordinary wet spinning method and going through a scouring stepand drying step to obtain aromatic fibers.

Granules

(1) Sucralose 0.1 (2) Filaggrin gene expression promoting agent 0.1 ofpresent invention: Zanthoxylum extract (3) Flavoring agent 5.0 (4)Excipient (Ceolus) 10.0 (5) Maltitol Balance

Tablets (Chewable)

(1) Inositol 11.0 (2) Maltitol 21.0 (3) Sucrose 0.5 (4) Salmon miltextract (DNA Na) 0.1 (5) Yeast extract 0.1 (6) Filaggrin gene expressionpromoting agent 0.1 of present invention: Basil oil (7) Flavoring agent5.0 (8) Excipient Balance

Tablets

(1) Lubricant (such as sucrose fatty acid ester) 1.0 (2) Aqueous gumarabic solution (5%) 2.0 (3) Acidifier 1.0 (4) Colorant As suitable (5)Filaggrin gene expression promoting agent 0.1 of present invention:Pepper oil (6) Sugar (such as powdered sugar or sorbitol) Balance

Candy

(1) Sugar 50.0 (2) Starch syrup 47.95 (3) Organic acid 2.0 (4) Filaggringene expression promoting agent 0.05 of present invention: Rose oil

Gum

(1) Sugar 43.0 (2) Gum base 30.95 (3) Glucose 10.0 (4) Starch syrup 16.0(5) Filaggrin gene expression promoting agent 0.05 of present invention:Basil oil

Cleansing Lotion

(1) 1,3-butylene glycol 6.0 (2) Glycerin 4.0 (3) Oleyl alcohol 0.1 (4)POE(20) sorbitan monolaurate 0.5 (5) POE(15) lauryl alcohol ester 0.5(6) Ethanol 10.0 (7) Filaggrin gene expression promoting agent 1.0 ofpresent invention: 3-(1′-piperidine) propionic acid (8) Purified waterBalance

Milky Lotion

(1) Microcrystalline wax 1.0 (2) Beeswax 2.0 (3) Lanolin 20.0 (4) Liquidparaffin 10.0 (5) Squalane 5.0 (6) POE(20) sorbitan monooleate 1.0 (7)Propylene glycol 7.0 (8) Filaggrin gene expression promoting agent 0.5of present invention: 3-(1′-piperidine) propionic acid (9) Filaggringene expression promoting agent 0.1 of present invention: Zanthoxylumextract (10) Sodium bisulfite 0.01 (11) Ethyl 4-hydroxybenzoate 0.3 (12)Fragrance As suitable (13) Purified water Balance

The invention claimed is:
 1. A cosmetic method for promoting expressionof filaggrin gene, comprising: administering a filaggrin gene expressionpromoting agent containing zanthoxylum extract and3-(1′-piperidine)propionic acid, to a subject desiring promotion of theexpression of the filaggrin gene.
 2. The cosmetic method according toclaim 1, wherein the filaggrin gene expression promoting agent consistsessentially of zanthoxylum extract and 3-(1′-piperidine) propionic acid.3. The cosmetic method according to claim 1, wherein the filaggrin geneexpression promoting agent contains 0.1% zanthoxylum extract and 0.1%3-(1′-piperidine) propionic acid.
 4. The cosmetic method according toclaim 1, wherein the subject further desires enhancing skinmoisturizing.
 5. The cosmetic method according to claim 4, wherein saidadministering results in enhancing of the skin moisturizing in thesubject through enhancing the filaggrin gene expression in the subject.6. The cosmetic method according to claim 1, wherein the subject furtherdesires enhancing a skin barrier function.
 7. The cosmetic methodaccording to claim 1, wherein said administering results in enhancing ofthe skin barrier function in the subject through enhancing the filaggringene expression in the subject.
 8. The cosmetic method according toclaim 1, wherein the subject further desires enhancing skin moisturizingand skin barrier function.
 9. The cosmetic method according to claim 1,wherein said administering results in enhancing of the skin barrierfunction in the subject through enhancing the filaggrin gene expressionin the subject.
 10. The cosmetic method according to claim 1, whereinsaid filaggrin gene expression promoting agent is administered to thesubject at a time at which the biological rhythm of filaggrin geneexpression level and the rhythm of the filaggrin gene expression levelresulting from administration of the filaggrin gene expression promotingagent are synchronous.
 11. The cosmetic method according to claim 2,wherein the subject further desires enhancing skin moisturizing.
 12. Thecosmetic method according to claim 11, wherein said administeringresults in enhancing of the skin moisturizing in the subject throughenhancing the filaggrin gene expression in the subject.
 13. The cosmeticmethod according to claim 2, wherein the subject further desiresenhancing a skin barrier function.
 14. The cosmetic method according toclaim 13, wherein said administering results in enhancing of the skinbarrier function in the subject through enhancing the filaggrin geneexpression in the subject.
 15. The cosmetic method according to claim 2,wherein the subject further desires enhancing skin moisturizing and skinbarrier function.
 16. The cosmetic method according to claim 15, whereinsaid administering results in enhancing of the skin barrier function inthe subject through enhancing the filaggrin gene expression in thesubject.
 17. The cosmetic method according to claim 3, wherein thesubject further desires enhancing skin moisturizing.
 18. The cosmeticmethod according to claim 17, wherein said administering results inenhancing of the skin moisturizing in the subject through enhancing thefilaggrin gene expression in the subject.
 19. The cosmetic methodaccording to claim 3, wherein the subject further desires enhancing askin barrier function.
 20. The cosmetic method according to claim 19,wherein said administering results in enhancing of the skin barrierfunction in the subject through enhancing the filaggrin gene expressionin the subject.